Biomimetic modeling of the first substrate reaction at the active site of ribonucleotide reductases. Abstraction of H3' by a thiyl free radical

Full text of DOI:10.1021/ja984399r

J. Am. Chem. Soc. 1999, 121, 5823-5824
5823
control nitrate ester 11) with a phenylsulfinyl radical leaving
Biomimetic Modeling of the First Substrate Reaction
at the Active Site of Ribonucleotide Reductases.
Abstraction of H3′ by a Thiyl Free Radical1
group7 at C2 to more closely model the enzyme process.
Morris J. Robins* and Gregory J. Ewing
Department of Chemistry and Biochemistry
Brigham Young UniVersity, ProVo, Utah 84602-5700
ReceiVed December 22, 1998
We have obtained the first conclusiVe chemical evidence
consistent with the controversial first two steps in the mechanism
postulated to occur during reductive deoxygenation at the active
site of ribonucleotide reductases.2,3 Generation of a proximal
primary aliphatic thiyl radical in a tetrahydrofuran model sub-
stituted at C2 with a radical leaving group results in abstraction
of H3 and elimination of phenylsulfinyl from C2.
Ribonucleotide reductases catalyze the deoxygenative reduction
of ribonucleoside 5′-(di or tri)phosphates to 2′-deoxynucleotides
and provide the only de novo source of DNA components. The
ribonucleoside 5′-diphosphate reductase (RDPR) from Escherichia
coli has been studied extensively.4 Its R1 homodimer subunit
contains substrate and allosteric binding sites and cysteine residues
required for catalysis. The R2 homodimer contains a diiron chelate
and an essential tyrosyl free radical.2-5 Mammalian and certain
viral-encoded RDPRs are similar. A postulated radical-cascade
mechanism for substrate reduction invokes long-range electron
transfer between •OTyr122 (in R2) and Cys439 (in R1) at the
active site interface. The •SCys439 radical generated in proximity
with the â face of the substrate is proposed2,3 to abstract the 3′-
hydrogen atom as the first substrate-activation step (Scheme 13).
Abstraction of H3′ from 1 by a primary aliphatic thiyl radical to
generate 2 has aroused debate,2e owing to the absence of an
appropriate chemical precedent.
We have shown that aminyl or oxyl radicals at C6 of
hexofuranosyl models abstract H3 by a [1,5]-hydrogen shift.1,6
Treatment (Bu3SnD/AIBN/benzene/∆) of 6-azido or 6-O-nitro
precursors produces C3 radicals that undergo deuterium transfer
from the stannane to give 3-[2H] product(s). The absence of (H
f D) exchange at C3 with a 6-S• radical in models that operate
with 6-O• (60-80%) or 6-N• (∼20%) radicals was troubling.1
However, RDPR executes abstraction of H3′ and a [1,2]-electron
shift coupled with hydrogen transfer from O3′ to O2′ and
“irreversible” loss of water from C2′ to give the stabilized oxallyl
radical 3 (Scheme 1).3 We now have synthesized thioether 5 (and
Bu3SnH (2 equiv)/AIBN (2 equiv)/benzene was added (24 h,
syringe pump) to a refluxing solution of 5 (Scheme 2) in benzene.
The concentrated mixture contained (1H NMR) vinyl ether 9 and
3-O-methyl-2-(phenylsulfinyl)-containing products (∼2:3). Chro-
matography gave the somewhat unstable and volatile dihydrofuran
9 (21%) and 1,4-anhydro-2,5-dideoxy-3-O-methyl-2-(phenyl-
sulfinyl)-6-thio-D-ribo-hexofuranitol (10, 52%). Formation of 9
and 10 is consistent with attack of a tributylstannyl radical on 5
to give 6, followed by double homolytic â-elimination to generate
thiyl radical 7 and ethylene. Intramolecular [1,5]-hydrogen transfer
of H3 to the 6-thiyl radical and elimination of phenylsulfinyl
radical from 8 would produce 9. Coupling of 7 with tributylstannyl
radical, and S-Sn bond cleavage upon chromatography, would
give 10. Conversion of 7 to 9 represents the first “relevant”
biomimetic modeling of the proposed abstraction of H3′ from
C3′ of ribonucleotides by •SCys439 of RDPR.
Formation of 6-S• radicals is assured by indirect Barton-
Robins8 generation via S-{2-[(phenoxythiocarbonyl)oxy]ethyl}
group removal, and high dilution reduced rates of bimolecular
coupling of tributylstannyl and thiyl radicals. Furan models
minimize steric/stereoelectronic effects at C1 and preclude radical
coupling with nucleobases (C8 of adenine, C6 of uracil).9 The
6-O-nitro ester 11 served as a positive control with demonstrated
ability to abstract H3 (via generation of a 6-O• radical),1,6 and
the sulfoxide 11 was thermally stable in refluxing benzene for
72 h.
Addition (5 h, syringe pump) of Bu3SnH (2 equiv)/AIBN (2
equiv)/benzene to a refluxing solution of 11 in benzene resulted
in exclusive formation of vinyl ether 14. Benzoylation of the
somewhat volatile 14 gave 15 (69% from 11). Formation of 14
is consistent with abstraction of H3 (1,5-shift) to the 6-oxyl radical
of 12 and â-elimination of phenylsulfinyl from 13.1,6 The stability
of the phenylsulfinyl radical precludes its participation in chain
reactions involving Bu3SnH. Stoichiometric quantities of initiator
were required, and only trace formation of 14 was observed with
10-15% molar ratios of AIBN.
The abstraction of H3′ from C3′ by •SCys439 has remained
controversial,2e and it is often assumed that thiyl radicals are poor
hydrogen abstractors because thiols are excellent hydrogen donors.
However, the chemistry of sulfur radicals is complex.10 Bond
dissociation energies for certain RS-H and RR′(HO)C-H
systems are similar,2e,11 but rates of hydrogen abstraction by thiyl
radicals (RH + •SR f •R + HSR) are generally ∼104 slower
than the reverse donation of hydrogen to alkyl radicals by
thiols.11,12
(1) Nucleic Acid Related Compounds. 109. Paper 108 is: Guo, Z.; Samano,
M. C.; Krzykawski, J. W.; Wnuk, S. F.; Ewing, G. J.; Robins, M. J.
Tetrahedron 1999, 55, 5705-5718.
(2) (a) Stubbe, J. AdV. Enzymol. Relat. Areas Mol. Biol. 1989, 63, 349-
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(d) Stubbe, J.; van der Donk, W. A. Chem. Biol. 1995, 2, 793-801. (e) Stubbe,
J.; van der Donk, W. A. Chem. ReV. 1998, 98, 705-762.
(3) Siegbahn, P. E. M. J. Am. Chem. Soc. 1998, 120, 8417-8429.
(4) (a) Thelander, L.; Reichard, P. Annu. ReV. Biochem. 1979, 48, 133-
158. (b) Reichard, P. Science 1993, 260, 1773-1777.
(5) (a) Nordlund, P.; Sjo¨berg, B.-M.; Eklund, H. Nature 1990, 345, 593-
598. (b) Uhlin, U.; Eklund, H. Nature 1994, 370, 533-539. (c) Sjo¨berg, B.-
M. In Nucleic Acids and Molecular Biology; Eckstein, F., Lilley, D. M. J.,
Eds.; Springer-Verlag: Berlin, 1995; Vol. 9, pp 192-221. (d) Robins, M. J.;
Samano, M. C.; Samano, V. Nucleosides Nucleotides 1995, 14, 485-493. (e)
Uhlin, U.; Eklund, H. J. Mol. Biol. 1996, 262, 358-369. (f) Kauppi, B.;
Nielsen, B. B.; Ramaswamy, S.; Larsen, I. K.; Thelander, M.; Thelander, L.;
Eklund, H. J. Mol. Biol. 1996, 262, 706-720. (g) Logan, D. T.; Su, X.-D.;
A° berg, A.; Regnstro¨m, K.; Hajdu, J.; Eklund, H.; Nordlund, P. Structure 1996,
4, 1053-1064. (h) Eriksson, M.; Uhlin, U.; Ramaswamy, S.; Ekberg, M.;
Regnstro¨m, K.; Sjo¨berg, B.-M.; Eklund, H. Structure 1997, 5, 1077-1092.
(i) Robins, M. J. Nucleosides Nucleotides 1999, 18, 779-793.
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10.1021/ja984399r CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/05/1999

5824 J. Am. Chem. Soc., Vol. 121, No. 24, 1999
Communications to the Editor
Scheme 1a
a Early steps of the Stubbe-Siegbahn mechanism for substrate reduction with RDPR.2,3
Scheme 2a
thiyl radicals such as •SSiPh3 efficiently catalyze the racemization
of (R)-2-(acetoxymethyl)tetrahydrofuran (via R-hydrogen abstrac-
tion), whereas a •SCys(OMe) model was “totally ineffective” as
a catalyst.14b Thus, hydrogen abstraction by thiols in isolated
models, taken out of context of the aliphatic cysteinyl radical
abstraction of a proximal secondary carbinol hydrogen on a
furanosyl ring,2e does not provide compelling evidence for such
a process at the enzyme active site.
In summary, we have prepared 5 and demonstrated hydrogen
abstraction from C3 by an aliphatic thiyl radical with accompany-
ing elimination of phenylsulfinyl radical from C2 to generate 9
(and a parallel positive control with oxyl radical generation from
11 to produce 14). This biomimetic modeling of abstraction of
H3′ by •SCys439 conclusiVely demonstrates the feasibility of step
1 in Scheme 1 for the first time. It is compatible with Siegbahn’s
calculations that indicate a “concerted” [1,2]-electron shift from
C3′ to C2′, hydrogen shift from O3′ to O2′, and elimination of
hydrogen-bonded water from C2′ with minimal charge separation.3
Hydrogen transfer from Cys225 to C2′ at the R-face of 3 produces
the 2′-deoxy-3′-ketonucleotide intermediate 4 in the reductive
deoxygenation sequence executed by the remarkable RDPR.
Acknowledgment. We thank the American Cancer Society (DHP-
34) and Brigham Young University development funds for support, Dr.
M. A. Peterson for use of a syringe pump, and Mrs. Jeanny K. Gordon
for assistance with the manuscript.
a (a) Bu3SnH/AIBN/benzene/∆. (b) BzCl/DMAP/pyridine/CH2Cl2.
It was shown in 1966 that treatment of â-hydroxy thioethers
with thiyl radicals gave thiols and ketones,13 and evidence for
hydrogen abstraction by thiyl radicals has been noted.2e,10-14
However, Roberts recently demonstrated that highly electrophilic
Supporting Information Available: Synthesis (Scheme 3) and
experimental details with characterization and spectral data (PDF). This
material is available free of charge via the Internet at http://pubs.acs.org.
JA984399R
(12) Scho¨neich, C.; Bonifacic, M.; Dillinger, U.; Asmus, K.-D. In ref 10b,
pp 367-376.
(13) Huyser, E. S.; Kellogg, R. M. J. Org. Chem. 1966, 31, 3366-3369.
(14) (a) Dang, H.-S.; Roberts, B. P. J. Chem. Soc., Perkin Trans.1 1998,
67-75. (b) Cai, Y.; Roberts, B. P. Chem. Commun. 1998, 1145-1146.